Ureidoalkyl substituted tetrazole thiol intermediates for preparing cephalosporins

ABSTRACT

The uriedoalkyl substituted tetrazolethiol compounds of this invention are used for preparing 7-acyl-3-(uriedoalkyl substituted tetrazolythiomethyl)-cephalosporins.

This is a division of application Ser. No. 639,033 filed Dec. 9, 1975,now U.S. Pat. No. 4,025,626.

This invention relates to a new series of cephalosporin compounds whichhave antibacterial activity when administered parenterally and orallyand to intermediates for the preparation thereof. In particular, thestructures of the biologically active cephalosporin compounds of thisinvention are characterized by having an ureidoalkyl substitutedtetrazolylthiomethyl group at the 3-position of the cephem nucleus.Also, this invention extends to methods and compositions for treatingcertain bacterial infections using these new compounds as well as tocertain chemical intermediates and methods for preparing the compoundsdescribed hereafter.

The compounds of this invention are represented by the followingstructural formula: ##STR1## in which:

R¹ is an acyl group selected from the group consisting of: ##STR2##where:

X is thienyl, dihydrophenyl, phenyl or phenyl monosubstituted withhydroxy, hydroxymethyl, formamido, ureido or carboxymethylamino;

A is NH₂, OH, COOH or SO₃ H; or formyloxy when X is phenyl;

Y is cyano, aminomethylphenyl, sydnone, pyridone, thienyl or tetrazolyl;

Z is methyl, trifluoromethyl, trifluoroethyl, pyridyl or cyanomethyl;

M IS ZERO TO TWO; AND

N IS TWO TO FIVE,

Or a non-toxic pharmaceutically acceptable salt thereof.

It will be recognized that the 4-carboxylic acid group of the compoundsof Formula I may be readily esterified by methods well known to the art.These esters include, for example, simple alkyl and aryl esters as wellas esters which are easily cleaved, within the body, to the parent acidsuch as indanyl, pivaloyloxymethyl, acetoxymethyl, propionyloxymethyl,glycyloxymethyl, phenylglycyloxymethyl and thienylglycyloxymethyl estersand others. Of course, when A is COOH, this group may be similarlyesterified. All such esters are included within the scope of thisinvention.

Preferred compounds of this invention are represented by Formula I wheren is two.

Advantageous compounds of this invention are represented by Formula Iwhere n is two and R¹ is ##STR3##

Most advantageous are the compounds represented by formula I where n istwo, R¹ is ##STR4## X is phenyl or hydroxyphenyl and A is NH₂ or OH.

Examples of the most preferred 7-acyl substituents (R¹ NH--) of thecompounds of Formula I are listed below:

α-hydroxyphenylacetamido

α-aminophenylacetamido

α-amino-4-hydroxyphenylacetamido

trifluoromethylthioacetamido

methylthioacetamido

2,2,2-trifluoroethylsulfinylacetamido

cyanoacetamido

α-carboxythienylacetamido

α-carboxyphenylacetamido

α-sulfophenylacetamido

methylsulfonylacetamido

cyanomethylthioacetamido

α-amino-4-carboxymethylaminophenylacetamido

2-aminomethylphenylacetamido

3-sydnoneacetamido

1-tetrazolylacetamido

2-thienylacetamido

2-pyridoneacetamido

4-pyridoneacetamido

4-pyridylthioacetamido.

Particularly preferred is the compound7-D-mandelamido-3-[1-(2-ureidoethyl)tetrazol-5-ylthiomethyl]-3-cephem-4-carboxylicacid.

Cephalosporin derivatives having 7-acyl substituents as defined aboveare well documented in the prior art. Substitution by variouslysubstituted S-heterocycliothiomethyl groups (--CH₂ SHet) at the 3position of the cephem nucleus is also known. Belgian Patent No. 823,861generically discloses7-[2-(2-exo-substituted-4-thiazolin-4-yl)acetamido]cephalosporinshaving, among others, a tetrazolylthiomethyl group at the 3-positionwhich may be substituted by, inter alia, a N-carbamoyl substitutedaminoalkyl group. No examples or other specific disclosure of anycompound containing such a N-carbamoyl substituted aminoalkylsubstituent on any 3-heterocyclic thiomethyl group are present in thereference. No other references to cephalosporin compounds containing the3-(ureidoalkyl substituted tetrazolyl)thiomethyl moiety disclosed hereinare believed to be known to the art.

The compounds of Formula I are prepared by acylation of an appropriate7-amino-3-ureidoalkyltetrazolylthiomethyl cephalosporin nucleus ofFormula II: ##STR5## in which:

n is two to five; and

R² is hydrogen or a protecting ester group, with an appropriateacylating agent followed by removal of the protective groups whenpresent. The carboxylic acid group of the acylating agent is activatedby any of the standard methods such as conversion to the mixedanhydride, acid chloride, acid imidazolide or activated ester. Inaddition, a reagent such as dicyclohexylcarbodiimide can be usedprovided that the carboxyl group on the cephem nucleus is protected withan easily removable protecting group such as a benzhydryl, t-butyl,trichloroethyl, benzyl, benzyloxymethyl, p-methoxybenzyl orp-nitrobenzyl ester. When A is NH₂, the α-amino group of the acylatingagent is, preferably, protected prior to acylation with an easilyremovable protective group known in the art such as t-butoxycarbonyl,trichloroethoxycarbonyl, benzyloxycarbonyl, the methyl acetoacetateadduct or similar groups commonly used in the synthesis of peptides. Thecompounds represented by Formula II above are also considered as objectsof this invention.

Alternatively, the compounds of Formula I are prepared by acylating7-aminocephalosporanic acid with an appropriately protected acylatingagent, as described above, and then displacing the 3-acetoxy group withthe desired ureidoalkyltetrazole thiol with subsequent removal of theprotective group(s). The ureidoalkyltetrazole thiols of the formula:##STR6## in which n is two to five, are also objects of this invention,being important intermediates for producing pharmaceutical end productsas described herein.

The protective groups can be removed according to methods well known tothe art, such as with trifluoroacetic acid when t-butyl ort-butoxycarbonyl protective groups are used. The resulting salt isconverted to the zwitterionic product or to the free acid by means of abasic ion exchange resin such as polystyrene-amine ion exchange resin(Amberlite IR-45) or else by basification of an aqueous solution of thesalt.

The acylating agents used as starting materials are either known orprepared by known methods.

The 7-amino-3-ureidoalkyltetrazolylthiomethyl cephalosporin startingmaterials of Formula II are prepared by reaction of7-aminocephalosporanic acid and an ureidoalkyltetrazole thiol of FormulaIII and then esterified.

The ureidoalkyltetrazole thiols of Formula III are prepared by reactionof the corresponding 1-aminoalkyl-5-(2,4-dinitrophenylthio)tetrazolecompounds, prepared from 2,4-dinitrofluorobenzene and an1-acetamidoalkyltetrazole-5-thiol followed by acid hydrolysis of theacetamido moiety with cyanic acid, which is prepared for example frompotassium cyanate and acetic acid, with subsequent cleavage of the2,4-dinitrophenyl protecting group. The1-acetamidoalkyltetrazole-5-thiols are prepared by reaction of anacetamidoalkyldithiocarbamate such as methyl2-acetamidoethyldithiocarbamate with an azide such as sodium azide. Theacetamidoalkyldithiocarbamates are prepared by treatment of anN-aminoalkylacetamide such as N-(2-aminoethyl)acetamide with carbondisulfide and an alkyl halide such as methyl iodide in the presence of abase such as triethylamine.

Certain compounds of this invention are capable of forming salts with,for example, the alkali metals such as sodium or potassium, the alkalineearth metals such as calcium or with the ammonium cation. When A is NH₂,the compounds can exist as the zwitterion or as either an acid or basesalt. These salts are prepared by standard methods using a wide varietyof non-toxic pharmaceutically acceptable acids and bases known in theart and are also considered as objects of this invention.

It will be recognized that due to the asymmetric α-carbon atom in the7-acetamido group of Formula I when R¹ is ##STR7## optical isomers willexist. Racemic or resolved products are obtained depending upon whethera racemic or resolved sidechain acid is used as an acylating agent. Theresolved sidechain acids are readily obtained from the racemic compoundsby resolution according to well known methods, including fractionalcrystallization of a salt formed with an optically active acid or base.All of the isomers, including separated isomers and mixtures thereof,are included within the scope of this invention.

The compounds of Formula I have exceptional antibacterial activityagainst both Gram-positive and Gram-negative organisms. Minimuminhibitory concentrations (MIC's) range from 0.1 to >200 μg./ml. in invitro testing. Test results for the compound7-D-mandelamido-3-[1-(2-ureidoethyl)tetrazol-5-ylthiomethyl]-3-cephem-4-carboxylicacid are given below:

    ______________________________________                                        Bacteria            MIC (μg./ml.)                                          ______________________________________                                        S. aureus HH 127    3.1, 1.6                                                  S. aureus SK 23390  0.4, 0.4                                                  S. villaluz SK 70390                                                                              25, 25                                                    Strep. faecalis HH 34358                                                                          25, 25                                                    E. coli SK 12140    0.8, 0.8                                                  E. coli HH 33779    1.6, 1.6                                                  Kleb. pneumo. SK 4200                                                                             0.8, 0.8                                                  Kleb. pneumo. SK 1200                                                                             0.4, 0.4                                                  Salmonella ATCC 12176                                                                             0.8, 0.2                                                  Shigella HH 117     0.1, 0.2                                                  Pseudo aerug. HH 63 >200, >200                                                Serratia Marc. ATCC 13880                                                                         25, 25                                                    Proteus morgani 179 0.8, 0.8                                                  Entero aerog. ATCC 13048                                                                          1.6, 6.3                                                  Entero. cloacae HH 31254                                                                          0.8, 0.8                                                  ______________________________________                                    

In the in vivo mouse protection test,7-D-mandelamido-3-[1-(2-ureidoethyl)tetrazol-5-ylthiomethyl]-3-cephem-4-carboxylicacid exhibited ED₅₀ 's of 0.86 mg./kg. against E. coli 12140 and 1.34mg./kg. against Kleb. pneumo. 4200 upon subcutaneous injection; and 17.5mg./kg. against E. coli 12140 and 25.5 mg./kg. against Kleb. pneumo.4200 upon oral administration.

Pharmaceutical compositions having antibacterial activity which comprisea pharmaceutical carrier containing an active but non-toxic quantity ofa compound of Formula I as well as methods of combatting bacterialinfections by administering such a composition to an infected host in anon-toxic amount sufficient to combat such infections are also objectsof this invention. The administration may be orally or by parenteralinjection such as subcutaneously, intramuscularly or intravenously. Theinjection of suitably prepared sterile solutions or suspensionscontaining an effective, non-toxic amount of the new cephalosporincompound is the preferred route of administration.

The compounds of Formula I are formulated and administered in the samemanner as other cephalosporins. The dosage regimen comprisesadministration, preferably by injection, of an active but non-toxicquantity of a compound of Formula I selected from the dosage unit rangeof from 100 to 1000 mg. with the total daily dosage regimen being from400 mg. to 6 g. The precise dosages are dependent upon the age andweight of the subject and on the infection being treated and can bedetermined by those skilled in the art based on the data disclosedherein compared with that available to the art attained with knowncephalosporins.

The following examples illustrate the invention but are not to beconstrued as limiting the scope thereof. Temperatures are in degreesCentigrade (° C.) unless otherwise stated.

EXAMPLE 17-D-Mandelamido-3-[1-(2-ureidoethyl)tetrazol-5-ylthiomethyl]-3-cephem-4-carboxylicacid

To a solution of 20.4 g. (0.20 mol.) of N-(2-aminoethyl)acetamide in 200ml. of 95% ethanol was added 27.9 ml. (0.20 mol.) of triethylamine and12.0 ml. (0.20 mol.) of carbon disulfide. The exothermic reactionreached reflux and then was cooled to ambient temperature over a 1.5hour period. Methyl iodide (28.4 g., 0.20 mol.) was added which againproduced an exothermic reaction. After 1.75 hours the reaction mixturewas evaporated to dryness and the solid residue was dissolved in 200 ml.of water. The aqueous solution was extracted twice with 250 ml. portionsof ethyl acetate. The extracts were combined, shaken with sodiumthiosulfate, dried (MgSO₄) and evaporated to dryness to give methyl2-acetamidoethyldithiocarbamate.

To a solution of 38.4 g. (0.198 mol.) of methyl2-acetamidoethyldithiocarbamate in 100 ml. of 95% ethanol was added asolution of 13.5 g. (0.208 mol.) of sodium azide in 100 ml. of water.The reaction mixture was refluxed for 24 hours then cooled andconcentrated under reduced pressure to about half volume. The solutionwas cooled to 15°, 50 ml. of 6N sulfuric acid was added and the acidicsolution was filtered. The filtrate was concentrated to about 100 ml.,chilled at 5° and 1-(2-acetamidoethyl)tetrazole-5-thiol was collected,m.p. 139°-139.5°. Additional amounts of the product were obtained bycontinuous extraction of the filtrate with ethyl acetate.

A solution of 9.3 g. (0.050 mol.) of 2,4-dinitrofluorobenzene in 50 ml.of acetone was added to a solution of 9.35 g. (0.050 mol.) of1-(2-acetamidoethyl)tetrazole-5-thiol and 6.85 ml. (0.050 mol.) oftriethylamine in 100 ml. of acetone and the reaction mixture was stirredfor 1 hour. The solid was collected and recrystallized from acetonitrileto give 1-(2-acetamidoethyl)-5-(2,4-dinitrophenylthio)tetrazole, m.p.197°-198°.

A mixture of 6.5 g. (0.02 mol.) of1-(2-acetamidoethyl)-5-(2,4-dinitrophenylthio)tetrazole, 100 ml. of 12 Nhydrochloric acid and 100 ml. of 95% ethanol was refluxed for 4.5 hours.The mixture was evaporated to dryness to give a gummy residue whichcrystallized upon addition of ethanol to give1-(2-aminoethyl)-5-(2,4-dinitrophenylthio)tetrazole hydrochloride, m.p.217°-219° (d).

To a solution of 0.84 g. (0.010 mol.) of sodium bicarbonate and 0.81 g.(0.010 mol.) of potassium cyanate in 35 ml. of water containing 2.5 ml.of glacial acetic acid was added 3.15 g. (0.010 mol.) of1-(2-aminoethyl)-5-(2,4-dinitrophenylthio)tetrazole hydrochloride. Themixture was refluxed for 2.5 hours, then it was filtered and the solidproduct was washed with water and recrystallized from methanol-acetoneto give 5-(2,4-dinitrophenylthio)-1-(2-ureidoethyl)tetrazole, m.p.190°-191° (d).

A mixture of 10 g. (0.031 mol.) of5-(2,4-dinitrophenylthio)-1-(2-ureidoethyl)tetrazole and 60 ml. (0.055mol.) of 5% sodium methoxide in methanol was stirred for 2.5 hours. Anadditional 35 ml. (0.031 mol.) of sodium methoxide solution was addedand the mixture was stirred at 25° for 12 hours. Ether (ca. 2 L.) wasadded to the reaction mixture and the crystallized product wascollected, washed with ethyl acetate and recrystallized frommethanolether to give 1-(2-ureidoethyl)tetrazole-5-thiol sodium salt,m.p. 131°-134°.

C₄ H₇ N₆ OS . Na . 1 H₂ O Calculated: 21.05% C; 3.98% H; 36.83% N Found:20.97% C; 4.04% H; 36.76% N.

A solution of 1-(2-ureidoethyl)tetrazole-5-thiol sodium salt in water ispassed through an Amberlite IR-120H ion exchange resin column to give,after lyophilization, 1-(2-ureidoethyl)tetrazole-5-thiol.

A mixture of 1.5 g. (0.071 mol.) of 1-(2-ureidoethyl)tetrazole-5-thiolsodium salt and 2.14 g. (0.005 mol.) of 7-D-mandelamidocephalosporanicacid sodium salt in 25 ml. of water was heated at ca. 80° for 2.5 hours.The reaction mixture was passed through a XAD-8 resin column whileeluting with water and then methanol. The product-containing aqueousfractions were lyophilized to give7-D-mandelamido-3-[1-(2-ureidoethyl)tetrazol-5-ylthiomethyl]-3-cephem-4-carboxylicacid sodium salt hydrate.

C₂₀ H₂₁ N₈ O₆ S₂ . Na . 2.5 H₂ O Calculated: 39.89% C; 4.32% H; 18.61% NFound: 39.98% C; 3.86% H; 18.51% N.

An aqueous solution of7-D-mandelamido-3-[1-(2-ureidoethyl)tetrazol-5-ylthiomethyl]-3-cephem-4-carboxylicacid sodium salt is acidified with 3 N HCl to pH 2.5 and extracted withethyl acetate. The extract is dried over anhydrous magnesium sulfate andevaporated to dryness to give the title compound.

EXAMPLE 27-(D-α-Aminophenylacetamido)-3-[1-(2-ureidoethyl)tetrazol-5-ylthiomethyl]-3-cephem-4-carboxylicacid

A solution of 7.58 g. (0.015 mol.) of7-(D-α-t-butoxycarbonylaminophenylacetamido)cephalosporanic acid. 1.88g. (0.01 mol.) of 1-(2-ureidoethyl)tetrazole-5-thiol and 2.52 g. (0.03mol.) of sodium bicarbonate in 125 ml. of water is stirred at 60° for 5hours while maintaining the pH at 7.0-7.2 by addition of sodiumbicarbonate. The mixture is cooled and extracted with ethyl acetate. Theaqueous phase is acidified to pH 2.5 with 3N hydrochloric acid and theacidic solution is extracted again with ethyl acetate. The extract isdried (MgSO₄), filtered and evaporated to dryness to give7-(D-α-t-butoxycarbonylaminophenylacetamido)-3-[1-(2-ureidoethyl)tetrazol-5-ylthiomethyl]-3-cephem-4-carboxylicacid.

7-(D-α-Butoxycarbonylaminophenylacetamido)-3-[1-(2-ureidoethyl)tetrazol-5-ylthiomethyl]-3-cephem-4-carboxylicacid (ca. 1 g.) is stirred at 25° with 25 ml. of trifluoroacetic acidand 25 ml. of 1,3-dimethoxybenzene for 2.25 hours. The mixture isevaporated to dryness, ether is added to the residue and the precipitateis collected, washed with ether, stirred in acetonitrile for 2 hours,then collected and dried in vacuo to give the title compound as itstrifluoracetic acid salt. The salt is dissolved in water and thesolution is stirred with IR-45 ion exchange resin then lyophilized togive the title compound.

EXAMPLE 3

Reaction of the N-t-butoxycarbonyl derivative of the followingcephalosporanic acids:

7-(α-amino-4-hydroxyphenylacetamido)cephalosporanic acid

7-(α-amino-4-formamidophenylacetamido)cephalosporanic acid

7-(α-amino-3-formamidophenylacetamido)cephalosporanic acid

7-(α-amino-4-ureidophenylacetamido)cephalosporanic acid

7-(α-amino-3-ureidophenylacetamido)cephalosporanic acid

7-(α-amino-4-hydroxymethylphenylacetamido)cephalosporanic acid

7-(α-amino-1,4-cyclohexadienylacetamido)cephalosporanic acid

7-(α-amino-4-carboxymethylaminophenylacetamido)cephalosporanic acid

with 1-(2-ureidoethyl)tetrazole-5-thiol as described in the procedure ofExample 2 followed by removal of the protective group and conversion ofthe trifluoroacetic acid salt to the free acid as described thereingives the following compounds of this invention:

7-(α-amino-4-hydroxyphenylacetamido-3[1-(2-ureidoethyl)tetrazol-5-ylthiomethyl]-3-cephem-4-carboxylicacid

7-(α-amino-4-formamidophenylacetamido)-3-[1-(2-ureidoethyl)tetrazol-5-ylthiomethyl]-3-cephem-4-carboxylicacid

7-(α-amino-3-formamidophenylacetamido)-3-[1-(2-ureidoethyl)tetrazol-5-ylthiomethyl]-3-cephem-4-carboxylicacid

7-(α-amino-4-ureidophenylacetamido)-3-[1-2-ureidoethyl)tetrazol-5-ylthiomethyl]-3-cephem-4-carboxylicacid

7-(α-amino-3-ureidophenylacetamido-3-]1-(2-ureidoethyl)tetrazol5-ylthiomethyl]-3-cephem-4-carboxylic acid

7-(α-amino-4-hydroxymethylphenylacetamido)-3-[1-(2-ureidoethyl)tetrazol-5-ylthiomethyl]-3-cephem-4-carboxylicacid

7-(α-amino-1,4-cyclohexadienylacetamido)-3-[1-(2-ureidoethyl)tetrazol-5-ylthiomethyl]-3-cephem-4-carboxylicacid

7-(α-amino-4-carboxymethylaminophenylacetamido)-3-]1-(2-ureidoethyl)tetrazol-5-ylthiomethyl]-3-cephem-4-carboxylicacid.

EXAMPLE 4

7-(4-Hydroxymandelamido)-3-[1-(2-ureidoethyl)tetrazol-5-ylthiomethyl]-3-cephem-4-carboxylicacid is prepared by reaction of 7-(4-hydroxymandelamido)cephalosporanicacid sodium salt and 1-(2-ureidoethyl)tetrazole-5-thiol sodium salt asdescribed in the procedure of Example 1 followed by conversion of theproduct sodium salt to the free acid as described therein.

EXAMPLE 5

When the sodium salt of a cephalosporanic acid listed below:

7-(3-sydnoneacetamido)cephalosporanic acid

7-(2-thienylacetamido)cephalosporanic acid7-(1-tetrazolylacetamido)cephalosporanic acid is reacted with1-(2-ureidoethyl)tetrazole-5-thiol sodium salt by the proceduredescribed in Example 1 and the product is converted to the free acid asdescribed therein, the following compounds of this invention areobtained, respectively:

7-(3-syndnoneacetamido)-3-[1-(2-ureidoethyl)tetrazol-5-ylthiomethyl]-3-cephem-4-carboxylicacid

7-(2-thienylacetamido)-3-[1-(2-ureidoethyl)tetrazol-5-ylthiomethyl]-3-cephem-4-carboxylicacid7-(1-tetrazolylacetamido)-3-[1-(2-ureidoethyl)tetrazol-5-ylthiomethyl]-3-cephem-4-carboxylicacid.

EXAMPLE 6

7-(2-Aminomethylphenylacetamido)-3-[1-(2-ureidoethyl)tetrazol-5-ylthiomethyl]-3-cephem-4-carboxylicacid

When 7-(2-aminomethylphenylacetamido)cephalosporanic acid sodium salt isreacted with 1-(2-ureidoethyl)tetrazole-5-thiol sodium salt by theprocedure described in Example 1 and the product is converted to thefree acid as described therein, the title compound is obtained.

EXAMPLE 77-Trifluoromethylthioacetamido-3-[1-(2-ureidoethyl)tetrazol-5-ylthiomethyl]-3-cephem-4-carboxylicacid

A solution of 1.88 g. (10.0 mmol.) of1-(2-ureidoethyl)tetrazole-5-thiol, 0.840 g. of sodium bicarbonate and5.45 g. (12.5 mmol.) of 7-trifluoromethylthioacetamidocephalosporanicacid sodium salt in 60 ml of water is stirred at 70°-75° for 5 hourswhile maintaining the pH at 6.8 by addition of 5% aqueous sodiumcarbonate solution. The reaction mixture is cooled and diluted withwater. Ethyl acetate is added and the mixture is acidified to pH 2.0with 6N hydrochloric acid. The combined aqueous phases are furtherextracted with ethyl acetate and the extracts are dried (MgSO₄) andevaporated to dryness to give the title compound.

EXAMPLE 8

Reaction of the sodium salt of a cephalosporanic acid listed below:

7-(2,2,2-trifluoroethylthioacetamido)cephalosporanic acid

7-trifluoromethylsulfinylacetamidocephalosporanic acid with1-(2-ureidoethyl)tetrazole-5-thiol as described in the procedure ofExample 7 gives the following compounds of this invention as finalproducts:

7-(2,2,2-trifluoroethylthioacetamido)-3-[1-(2-ureidoethyl)tetrazol-5-ylthiomethyl]-3-cephem-4-carboxylicacid

7-trifluoromethylsulfinylacetamido-3-[1-(2-ureidoethyl)tetrazol-5-ylthiomethyl]-3-cephem-4-carboxylicacid.

EXAMPLE 97-Amino-3-[1-(2-ureidoethyl)tetrazol-5-ylthiomethyl]-3-cephem-4-carboxylicacid

A solution of 11.29 g. (0.06 mol.) of 1-(2-ureidoethyl)tetrazole-5-thiolin 120 ml. of acetone is added to a warm (45°) solution of 10.9 g. (0.04mol.) of 7-aminocephalosporanic acid in a mixture of 220 ml. of water,50 ml. of acetone and 8.4 g. (0.01 mol.) of sodium bicarbonate. Thetemperature is raised to 65° and the pH maintained at 7.4-7.6 byaddition of aqueous sodium carbonate solution. After 3 hours, theacetone is removed in vacuo and the reaction mixture is cooled to 10°and adjusted to pH 3.5 by addition of dilute hydrochloric acid. Theproduct is collected, washed with water and then acetone to give thetitle compound.

EXAMPLE 10

When an equivalent amount of an N-aminoalkylacetamide listed below:

N-(3-aminopropyl)acetamide

N-(4-aminobutyl)acetamide

N-(5-aminopentyl)acetamide

is used in the procedure of Example 1 in place ofN-(2-aminoethyl)acetamide and the resulting dithiocarbamates are treatedwith sodium azide to produce the corresponding1-acetamidoalkyltetrazole-5-thiols which are converted to the1-ureidoalkyl derivatives, all as described therein, the following1-ureidoalkyltetrazole-5-thiols are obtained:

1-(3-ureidopropyl)tetrazole-5-thiol

1-(4-ureidobutyl)tetrazole-5-thiol

1-(5-ureidopentyl)tetrazole-5-thiol.

Reaction of the sodium salt of a 1-ureidoalkyltetrazole-5-thiol listedabove with 7-D-mandelamidocephalosporanic acid sodium salt as describedin the procedure of Example 1 followed by conversion of the salt formedto the free acid, gives the following compounds of this invention:

7-D-mandelamido-3-[1-(3-ureidopropyl)tetrazol-5-ylthiomethyl]-3-cephem-4-carboxylicacid

7-D-mandelamido-3-[1-(4-ureidobutyl)tetrazol-5-ylthiomethyl]-3-cephem-4-carboxylicacid

7-D-mandelamido-3-[1-(5-ureidopentyl)tetrazol-5-ylthiomethyl]-3-cephem-4-carboxylicacid.

LIkewise, reaction of the substituted tetrazole thiols or thecorresponding sodium salts listed above with any of the 7-acylcephalosporanic acids mentioned herein or their corresponding saltsaccording to procedures described herein gives the correspondingcompounds of this invention.

EXAMPLE 117-(2,2,2-Trifluoroethylsulfinylacetamido)-3-[1-(2-ureidoethyl)tetrazol-5-ylthiomethyl]-3-cephem-4-carboxylicacid

To a stirred solution of 5.7 g. (0.3 mol.) of2,2,2-trifluoroethylsulfinylacetic acid and 3.45 g. (0.03 mol.) ofN-hydroxysuccinimide in 50 ml. of tetrahydrofuran at 0° is added 6.2 g.(0.031 mol.) of dicyclohexylcarbodiimide. The reaction mixture isstirred at 0° for 1 hour then at 25° for 12 hours. The precipitate isfiltered and washed with tetrahydrofuran and the filtrate is evaporatedto dryness to give the activated ester of2,2,2-trifluoroethylsulfinylacetic acid.

A suspension of 4.0 g. (0.01 mol.) of7-amino-3-[1-(2-ureidoethyl)tetrazol-5-ylthiomethyl]-3-cephem-4-carboxylicacid in 50 ml. of dry dimethylformamide is treated with 2 ml. oftriethylamine and the mixture is stirred for 15 minutes at 25°. A slightexcess of 0.01 mol. of the activated ester of2,2,2-trifluoroethylsulfinylacetic acid is added to the mixture and itis stirred an additional hour. The reaction mixture is evaporated todryness and water and ethyl acetate are added to the residue. The layersare separated, the ethyl acetate layer is discarded, fresh ethyl acetateis added to the aqueous phase and it is acidified to pH 2.5 by additionof 6N hydrochloric acid. The mixture is filtered, the layers areseparated and the aqueous phase is extracted with ethyl acetate. Thecombined extracts are washed with water, dried (MgSO₄) and evaporated todryness to give the title compound.

In like manner, the 7-(2,2,2-trifluoroethylsulfinylacetamido)derivatives of other 7-amino-3-ureidoalkyltetrazole cephalosporinsdescribed above may be prepared.

EXAMPLE 127-Methylthioacetamido-3-[1-(2-ureidoethyl)tetrazol-5-ylthiomethyl]-3-cephem-4-carboxylicacid

To a stirred, cooled (-20°) solution of 10.4 g. (0.026 mol.) of7-amino-3-[1-(2-ureidoethyl)tetrazol-5-ylthiomethyl]-3-cephem-4-carboxylicacid in 220 ml. of 3% sodium bicarbonate and 220 ml. of acetone is addeddropwise a solution of 3.66 g. (0.029 mol.) of methylthioacetyl chloridein 52 ml. of acetone, during which time the pH of the reaction mixtureis maintained at 8.0 by addition of 10% sodium hydroxide. After additionthe reaction mixture is stirred an additional 20 minutes at -15°, thenis warmed to 25° and extracted with ether. The remaining aqueous phaseis cooled, 250 ml. of ethyl acetate is added and the slurry is acidifiedwith 3N hydrochloric acid. The layers are separated and the aqueousphase is extracted twice more with ethyl acetate. The combined extractsare dried (MgSO₄) and evaporated to dryness to yield the title compound.

EXAMPLE 137-(D-α-Formyloxyphenylacetamido)-3-[1-(2-ureidoethyl)tetrazol-5-ylthiomethyl]-3-cephem-4-carboxylicacid

A mixture of 4.0 g. (0.01 mol.) of7-amino-3-[1-(2-ureidoethyl)tetrazol-5-ylthiomethyl]-3-cephem-4-carboxylicacid, 3.97 g. (0.02 mol.) of the formate ester of D-mandeloyl chlorideand 5 g. of sodium bicarbonate in 100 ml. of water and 140 ml. ofacetone is stirred in the cold for 1 hour, then at 25° for 2 hours. Theacetone is evaporated in vacuo and the remaining aqueous mixture isextracted with ethyl acetate. The aqueous solution is added withstirring to a cold mixture of 100 ml. of water and 200 ml. of ethylacetate and the pH of the resulting mixture is adjusted to 2.0 byaddition of 6N hydrochloric acid. The mixture is filtered, the layersare separated and the ethyl acetate layer is washed with water, dried(MgSO₄) and evaporated to dryness to give the title compound.

EXAMPLE 14

Acylation of7-amino-3-[1-(2-ureidoethyl)tetrazol-5-ylthiomethyl]-3-cephem-4-carboxylicacid with an activated derivative of an acid listed below:

cyanoacetic acid

cyanomethylthioacetic acid

4-pyridylthioacetic acid

2-pyridone-N-acetic acid

4-pyridone-N-acetic acid

as described in the procedure of Example 11 gives the followingcompounds of this invention:

7-cyanoacetamido-3-[1-(2-ureidoethyl)tetrazol-5-ylthiomethyl]-3-cephem-4-carboxylicacid

7-cyanomethylthioacetamido-3-[1-(2-ureidoethyl)tetrazol-5-ylthiomethyl]-3-cephem-4-carboxylicacid

7-(4-pyridylthioacetamido)-3-[1-(2-ureidoethyl)tetrazol-5-ylthiomethyl]-3-cephem-4-carboxylicacid

7-(2-pyridoneacetamido)-3-[1-(2-ureidoethyl)tetrazol-5-ylthiomethyl]-3-cephem-4-carboxylicacid

7-(4-pyridoneacetamido)-3-[1-(2-ureidoethyl)tetrazol-5-ylthiomethyl]-3-cephem-4-carboxylicacid.

EXAMPLE 15

Reaction of a cephalosporanic acid listed below or its correspondingsalt:

7-(α-hydroxy-2-thienylacetamido)cephalosporanic acid

7-(α-carboxy-2-thienylacetamido)cephalosporanic acid

7-(α-sulfophenylacetamido)cephalosporanic acid

with 1-(2-ureidoethyl)tetrazole-5-thiol sodium salt by proceduresdescribed hereinabove gives, after conversion of the product to the freeacid, the following compounds of this invention:

7-(α-hydroxy-2-thienylacetamido)-3-[1-(2-ureidoethyl)tetrazol-5-ylthiomethyl]-3-cephem-4-carboxylicacid

7-(α-carboxy-2-thienylacetamido)-3-[1-(2-ureidoethyl)tetrazol-5-ylthiomethyl]-3-cephem-4-carboxylicacid

7-(α-sulfophenylacetamido)-3-[1-(2-ureidoethyl)tetrazol-5-ylthiomethyl]-3-cephem-4-carboxylicacid.

EXAMPLE 167-(2,2,2-Trifluoroethylsulfonylacetamido)-3-[1-(2-ureidoethyl)tetrazol-5-ylthiomethyl]-3-cephem-4-carboxylicacid

To a solution of 8.6 g. (0.019 mol.) of7-amino-3-[1-(2-ureidoethyl)tetrazol-5-ylthiomethyl]-3-cephem-4-carboxylicacid t-butyl ester and 3.9 g. (0.019 mol.) of2,2,2-trifluoroethylsulfonylacetic acid in tetrahydrofuran is addeddropwise a solution of 3.9 g. (0.019 mol.) of dicyclohexylcarbodiimidein 100 ml. of tetrahydrofuran. The reaction mixture is stirred at 25°for 12 hours, then filtered and concentrated to about 10 ml. The residueis filtered and evaporated to dryness to give7-(2,2,2-trifluoroethylsulfonylacetamido)-3-[1-(2-ureidoethyl)tetrazol-5-ylthiomethyl]-3-cephem-4-carboxylicacid t-butyl ester.

The ester is dissolved in acetonitrile and trifluoroacetic acid isadded. The solution is stirred for 3 hours, then evaporated to drynessto give the title compound.

Likewise, 7-(2,2,2-trifluoroethylsulfonylacetamido) derivatives of theother 7-amino-3-substituted tetrazole cephalosporins disclosed hereinare prepared.

EXAMPLE 17

An injectable pharmaceutical composition is formed by adding sterilewater or sterile saline solution (2 ml.) to 500 mg. of7-D-mandelamido-3-[1-(2-ureidoethyl)tetrazol-5-ylthiomethyl]-3-cephem-4-carboxylicacid sodium salt.

Pharmaceutical compositions of the other antibacterial compoundsdisclosed above may be formulated in a similar manner.

EXAMPLE 18

A tablet or capsule is formed from 500 mg. of7-D-mandelamido-3-[1-(2-ureidoethyl)tetrazol-5-ylthiomethyl]-3-cephem-4-carboxylicacid, 250 mg. of lactose and 75 mg. of magnesium stearate.

Tablets or capsules of the other antibacterial compounds disclosed abovemay be formulated in a similar manner.

What is claimed is:
 1. A compound of the formula ##STR8## in which n istwo to five.
 2. A compound according to claim 1, said compound being1-(2-ureidoethyl)tetrazole-5-thiol.